Inflammatory Breast Cancer Metastasis Research Articles

Abstract P2-06-05: TP-0903, an AXL kinase inhibitor, reduces inflammatory breast cancer aggressiveness and macrophage polarization through additional mechanisms that may include JAK2 and Aurora B

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer; it accounts for 2-4% of breast cancer cases but causes 8-10% of breast cancer deaths. Novel targeted therapy to improve the outcomes of patients with IBC is urgently needed. The receptor tyrosine kinase AXL is a driver for metastasis and drug resistance in various cancers, including breast cancer. Our previous work showed that AXL signaling contributes to the aggressiveness of IBC. In addition, emerging evidence indicates that the tumor microenvironment components, particularly tumor-associated macrophages, are critical drivers of the IBC clinical phenotype and promote IBC metastasis. AXL signaling has been shown to modulate the tumor microenvironment. In the present study, we investigated the impact of TP-0903, a small-molecule AXL kinase inhibitor, with additional activity against Aurora B and Janus kinase 2 (JAK2), on IBC cells and macrophage polarization. Methods: The effects of TP-0903 on IBC cell proliferation, migration/invasion, and mammosphere formation were analyzed. The effects of TP-0903 on the polarization of human monocytic cells THP-1 were tested in vitro. In addition, the signaling pathways involved in TP-0903-regulated M2 macrophage polarization were investigated using Western blotting. Results: The half-maximal inhibitory concentration (IC50) of TP-0903 in an array of IBC cells (including SUM149, SUM190, BCX010, FC-IBC-02, MDA-IBC-3, and KPL4) ranged from 66 nM to 346 nM, suggesting a strong cell growth inhibitory effect. TP-0903 treatment decreased the migration, invasion, and mammosphere formation of IBC cells. In addition, TP-0903 inhibited both AXL signaling and Aurora B activation, which induced a G2/M cell cycle arrest in IBC cells. Based on the importance of AXL and JAK2 in the regulation of the tumor microenvironment, we showed that TP-0903 decreased expression of CD163/CD206 and the CCL17/CCL18 cytokine, and markers of M2 macrophages, suggesting that TP-0903 treatment inhibits the polarization of THP-1 cells to M2 macrophages in vitro. We also found that TP-0903 treatment decreased the phosphorylation of STAT6, a critical molecule in M2 polarization, and that knockdown of STAT6 expression decreased M2 macrophage polarization, indicating that TP-0903 may regulate macrophage polarization via STAT6 signaling. Conclusion: Our results demonstrated the dual functions of TP-0903 targeting of both IBC cells and macrophages, possibly via the targeting of multiple kinases, including AXL and Aurora B. Examinations of the impact of TP-0903 on the cross-talk between IBC cells and macrophages in vitro and in vivo and the related mechanisms are ongoing and will be presented at the meeting. Citation Format: Cheng Y, Funakoshi Y, Wang X, Warner SL, Bearss DJ, Ueno NT. TP-0903, an AXL kinase inhibitor, reduces inflammatory breast cancer aggressiveness and macrophage polarization through additional mechanisms that may include JAK2 and Aurora B [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-05.

CSF-1/CSF-1R axis is associated with epithelial/mesenchymal hybrid phenotype in epithelial-like inflammatory breast cancer

Inflammatory breast cancer (IBC) is a rare subtype of breast cancer, accounting for 8–10% of breast cancer-associated deaths in the US. Clinical hallmarks of IBC include tumor emboli in lymphatic vessels and E-cadherin overexpression, which supports a type of metastasis referred to as cell cluster-based metastasis, prevalent in IBC. In contrast, we previously reported epithelial-to-mesenchymal transition (EMT)-based progression of IBC, utilizing in vivo xenografts and in vitro Matrigel culture models. To address these two contradictory concepts of IBC metastasis, we used Matrigel culture to induce EMT in a panel of IBC cells. Results revealed Matrigel culture induced vimentin expression in SUM149 and SUM190 IBC cells at the transcriptional and protein levels while maintaining the expression of E-cadherin, a phenomenon referred to as partial EMT. Transcriptional profiling revealed that expression of colony-stimulating factor 1 (CSF-1) was induced in Matrigel culture. When the receptor tyrosine kinase of CSF-1 (CSF-1R) was inhibited by CSF-1R inhibitor BLZ945, the partial EMT was reversed in a dose-dependent manner, indicating that the CSF-1/CSF-1R axis plays a key role in controlling partial EMT. This observation may help reconcile the two contradictory theories of IBC metastasis, EMT vs cell cluster-based metastasis.

Abstract 2636: Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal form of primary breast cancer. Inflammatory signaling pathways are active in IBC, but the function of the immune response remains elusive. ErbB receptors play a role in IBC. ERBB2 is amplified in 50% of IBCs, and ERBB3 is also mutated in IBC. Currently, Lapatinib, a dual ErbB inhibitor, is used in IBC patients with ERBB2 amplification. Although immune cell inflammatory signaling may promote IBC tumor growth and metastasis, the presence of cytotoxic tumor-associated lymphocytes has also been associated with a more favorable breast cancer prognosis. The contradictory nature of the immune data highlights the need to elucidate the relationship between the immune response and subsequent treatments. Given the active IBC immune component and the recent clinical benefit of immune checkpoint inhibitors in cancer treatment, IBC’s may represent a clinically unique breast cancer population that may benefit from immunomodulating agents. Interestingly, Myc is a downstream effector of ErbB signaling, and Myc is thought to regulate the expression of immune checkpoint proteins CD47 and PD-L1. The presence of PD-L1-positive IBC immune infiltrate suggests that IBC’s may benefit from therapies that disrupt PD-L1 signaling together with ErbB inhibitors. METHODS: IHC was used to examine immune checkpoint signaling and characterize the tumor-immune infiltrate. Tumor tissues were also characterized using an RNA-seq panel that examined the expression of 377 immune-related genes. Cell lines (BT474, SKR3, AU565, and SUM225) were treated with Lapatinib and Neratinib to examine the relationship between growth factor receptors and downstream immune signaling pathways. RESULTS: RNA-seq revealed the expression of specific immunosuppressive signaling pathways in tumors. Treatment of breast cancer cells with ErbB inhibitors resulted in a decrease in the levels of PD-L1. Treatment with Lapatinib and Neratinib diminished PD-L1 in all cell lines. Levels of Phospho-Stat3 decreased in BT474 and Sum225 but not in SKBR3 and AU565, implying that PD-L1 is regulated by another mechanism (ERK-MYC). CONCLUSION: Our results provide mechanistic insight into ErbB receptor activation and the expression of downstream signaling molecules (Stat3, Myc, PD-L1, and others). In addition, our unique RNA-seq immune signature reveals the expression of several genes that may serve as biomarkers of inhibitory immune signaling pathways. Our laboratory is currently examining the correlation between PD-L1 expression and the activation of ErbB2 in model systems and clinical trials using ErbB inhibitors in combination with PD-L1 inhibitors. Our immune panel gene signature may serve as a useful diagnostic test that, in conjunction with traditional ErbB testing, can identify patients that will benefit from combination therapy of an ErbB inhibitor and an immune checkpoint inhibitor. Note: This abstract was not presented at the meeting. Citation Format: Christopher A. Hamm, Sumin Zhao, Cesar A. Santa-Maria, Massimo Cristofanilli, Neil L. Spector, Sarah Bacus. Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2636. doi:10.1158/1538-7445.AM2017-2636

Abstract 3302: High incidence of MAC387 positive cells in the carcinoma tissues of inflammatory breast cancer patients correlate with the detection of multiple human Cytomegalovirus genotypes and invasive properties of the disease

Abstract Introduction: Previously we showed that the incidence of multiple human cytomegalovirus (HCMV) genotypes in the carcinoma tissues of inflammatory breast cancer (IBC) patients plays essential role in the disease progression. Primary HCMV infection to monocytes induces differentiation and biological turnover of monocytes to macrophages. In addition infected macrophages serves as “mobile vectors” for virus spreading and dissemination to different organs mainly by transendothelial migration. In addition we screened for the infiltration of CD14+ and CD68+ monocytes/macrophages markers in the carcinoma tissues of IBC versus non-IBC patients we showed that of CD14+ cells highly infiltrate tumor microenvironment (TME) of IBC patients compared to non-IBC. Aims: In the present study we aim to 1) Assess the level of expression of MAC387 protein by monocytes/macrophages infiltrating TME of IBC versus non-IBC patients; 2) Test the correlation between the density of infiltrated MAC387+ cells and the incidence of different HCMV genotypes in carcinoma tissues of IBC versus non-IBC tissues. Since MAC387 found to be more common in cancers characterized by high metastatic properties we will also 3) determine whether the expression of MCA387 correlate with lymph-node metastasis and lymphovascular invasion in IBC versus non-IBC breast cancer patients. Materials and Methods: A total of 135 breast cancer patients (91 non-IBC and 44 IBC) were enrolled to the present study during the period of January 2012 to September 2015 from Ain Shams university Hospitals. Detection of MAC387 marker was assessed by immunohistochemistry and HCMV genotypes were detected using multiplex PCR methodology. Results: MAC387+ positive cells were more prevalent in IBC tissues than non-IBC tissues (p = 0.4). Incidence of higher number of MAC387+ cells were positively correlate with higher number of metastatic lymph nodes in both IBC and non-IBC patients r = 0.807 and 0.779 respectively. Moreover, Incidence of higher number of MAC387+ cells found to be positively correlate with lymphovascular invasion in IBC patients r = 0622. Detection of multiple HCMV genotypes was statistically higher (p = 0.04) in IBC tissues in comparison with non-IBC tissues. Moreover, triple negative non-IBC and IBC tissues showed higher incidence of multiple HCMV genotypes in comparison with hormonal positive non-IBC and IBC tissues. of the monocytes/macrophages MAC387+ positive cells were more prevalent in IBC tissues showed multiple HCMV genotypes in comparison with IBC tissues showed single HCMV genotype (p = 0.46). Conclusion: MAC387+ positive cells were more prevalent in IBC tissues and correlate with presence of multiple HCMV genotypes and high invasive properties of the disease. Citation Format: Hossam Taha Mohamed, Ramy Gadalla, Sherif Abdel Aziz Ibrahim, M. Akram Nouh, Mohamed El-Shinawi, Robert J. Schneider, Mona Mostafa Mohamed. High incidence of MAC387 positive cells in the carcinoma tissues of inflammatory breast cancer patients correlate with the detection of multiple human Cytomegalovirus genotypes and invasive properties of the disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3302.

Late pulmonary metastasis of inflammatory breast cancer 26 years after initial radiation therapy alone

A 64-year-old woman underwent cobalt-60 radiation therapy 26 years ago because of advanced inflammatory breast cancer. The initial stage was T4N0M0. During the course of cobalt-60 therapy, the patient presented severe chest wall necrosis. Her chest wall wound healed very slowly and she decided to stop radiation therapy and declined both surgery as well as chemotherapy. After chronic wound healing, the patient did not come back for follow-up. Twenty-six years later, she came back for mild dyspnea. Subsequent study showed suspicious lung metastasis. Later, she underwent thoracoscopic biopsy, with which lung metastasis was confirmed. She recovered well and currently receives oral chemotherapy for disease control.

A Preclinical Model of Inflammatory Breast Cancer to Study the Involvement of CXCR4 and ACKR3 in the Metastatic Process

Inflammatory breast cancer (IBC) is an aggressive and invasive tumor, accounting for 2.5% of all breast cancer cases, and characterized by rapid progression, regional and distant metastases, younger age of onset, and lower overall survival. Presently, there are no effective therapies against IBC and a paucity of model systems. Our aim was to develop a clinically relevant IBC model that would allow investigations on the role of chemokine receptors in IBC metastasis. Primary cultures of tumor cells were isolated from pleural exudates of an IBC patient and grown as spheres or monolayers. We developed a human xenograft model where patient-derived IBC cells, stably transduced with lentiviral vectors expressing fluorescent and bioluminescent markers, were inoculated directly into the left ventricle of mice. Our in vivo data show that these IBC cells (FC-IBC02A) are able to seed and proliferate into various organs, including brain, lungs, lymph nodes, and bone, closely replicating the metastatic spread observed in IBC patients. Moreover, cells were able to generate tumors when grafted in the mammary fat pad of mice. RT-PCR and microscopy studies revealed expression of both CXCR4 and ACKR3 receptors in FC-IBC02A cells. Furthermore, CXCL12 (the endogenous chemokine ligand of these receptors) induced transendothelial migration of these cells and stimulated signaling pathways involved in cell survival and migration - an effect reduced by CXCR4 or ACKR3 antagonists. This new model can be used to develop chemokine-based pharmacological approaches against the IBC metastatic process. This work also provides the first evidence of ACKR3 expression in IBC cells.

Abstract P5-01-04: In vivo lymphatic imaging of a human inflammatory breast cancer model

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive but poorly understood type of breast cancer. IBC constitutes only appoximately 5% of all newly diagnosed breast cancers, yet responsible for 8-10% of breast cancer-related deaths. Unfortunately, there are no definitive molecular or pathological, early diagnostic criteria for IBC. IBC grows in nests or sheets, spreading outward into the skin and eventually distant sites through dermal lymphatic vessels; therefore, one of the key pathological characteristics of IBC is dermal lymphatic invasion by tumor emboli, which can lead to obstruction of the lymphatic drainage possibly causing the clinical inflammatory features of diffuse erythema, rapid breast enlargement and edema as indicated by peau d’orange (orange peel) appearance covering at least a third of the breast surface. However, relatively little is known about the role of lymphatic function in IBC growth and metastasis. The purpose of this study was to non-invasively and longitudinally image changes of lymphatic drainage patterns in mice bearing the triple negative, human IBC SUM149 cells, which were stably transfected with iRFP gene reporter (iRFP-SUM149) in order to better monitor tumor growth and metastasis. Methods: Eight to ten weeks old female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were housed and fed special sterilized pelleted food and sterilized water. Two weeks after feeding special diets, mice were subcutaneously injected with iRFP-SUM149 cells in the left hindlimb or orthotopically in the left inguinal mammary fat pad (MFP). Fluorescence images were acquired immediately after and for up to 10 mins after i.d. injection of indocyanine green (ICG) using a custom-built NIRF imaging system. To achieve a greater magnification, a macrolens was used. Results: Our NIRF imaging data showed gradual changes of lymphatic vessels around the tumor. In mice with an orthotopic tumor, ICG accumulation was observed in the distal peritumoral region at up to 3 weeks p.i., indicative of the start of tumor obstruction of normal lymphatic drainage. As tumors grew, the greater extent of lymph flow, but not all, was obstructed by a tumor, resulting in dermal backflow and thus staining more lymphatic capillaries and eventually rerouting of lymphatic drainage was detected due to complete obstruction of lymph flow by a tumor. Similar to an orthotopic model, mice with a s.c. tumor also showed altered lymphatic drainage patterns during tumor growth. Interestingly, extravasation of ICG into iRFP-SUM149 was detected at 3 weeks p.i., due to leaky lymphatic vessels. A similar lymphatic phenotype has also been observed in IBC patients. Previously, we have imaged atypical tortuous lymphatics and altered lymphatic drainage in the arm and affected breast of a patient with IBC using clinical NIRF imaging developed in our team to image as deep as 3-5 cm with a microdose of ICG. Conclusions: Longitudinal, non-invasive imaging of lymphatic functional and architectural changes provides a new method to dynamically monitor lymphatic response to IBC growth and metastasis. Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Eva M Sevick-Muraca. In vivo lymphatic imaging of a human inflammatory breast cancer model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-04.

Abstract P6-14-06: CCR5 antagonists suppresses the migration and invasion of human inflammatory breast cancer cells

Abstract Background: Inflammatory breast cancer (IBC) is a rare and highly metastatic variant of breast cancer with a poor prognosis and lower survival rate. The disease has a peculiar pattern of early recurrence to soft tissue, bone and central nervous system irrespective of molecular subtype. Currently, there are no specific therapeutic options for IBC patients, particularly there are no therapeutic agents aimed at controlling metastatic spread. Chemokine CCL5 and its receptor CCR5 play a significant role in breast cancer progression and metastasis. We have recently shown that CCR5 promotes breast cancer invasiveness and metastatic potential, and CCR5 inhibition by the FDA approved CCR5 antagonists Maraviroc and Vicriviroc reduced in vivo metastasis in a basal-like breast cancer model (Cancer Res 2012;72:3839-3850). Here, we examined the expression of CCR5, as well as the effects of CCR5 inhibition on the migration and invasion in vitro in human IBC cells. Methods: IBC cell lines SUM149, SUM190, and FC-IBC-02 derived from pleural effusion fluid of an IBC patient were examined for the CCR5 expression by immunofluorescence staining. The effects of CCR5 antagonists (Maraviroc and Vicriviroc) on cell migration and invasion in vitro were examined using matrigel-coated Boyden chamber assay in FC-IBC-02 cells, which are triple-negative, basal-like, cancer stem cell phenotype, and rapidly developed primary tumors and metastasis in vivo (Breast Cancer Res Treat 2013;140:23–33). Cells were treated by Maraviroc and Vicriviroc at 100 µM. Results: CCR5 was expressed at low positive component comprising only about 5-7 % of the total cell population in SUM 149 cells. CCR5 expression was not detected in SUM190 cells. CCR5 was expressed at significantly higher levels with a higher percentage of positive population (50-60%) in FC-IBC-02 cells compared with SUM149 cells. FC-IBC-02 cells have a relatively high percentage of CCR5 positive cells in suspension culture. CCR5 inhibition by both CCR5 antagonists Maraviroc and Vicriviroc significantly inhibited the migration and invasion of IBC cells in vitro. Maraviroc and Vicriviroc demonstrated effective agents in controlling migrations (55% and 60% relative inhibition). Conclusions: CCR5 is highly expressed in human IBC cells. CCR5 antagonists demonstrated the inhibition of migration and invasion of IBC cells. Further studies are warranted to determine the effects of CCR5 antagonists in combination with standard treatment in the ability to control IBC progression and metastasis. These results may suggest a potential antimetastatic role for CCR5-inhibitors in IBC. Citation Format: Zhaomei Mu, Xuanmao Jiao, Richard G Pestell, Massimo Cristofanilli. CCR5 antagonists suppresses the migration and invasion of human inflammatory breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-06.

In vivo lymphatic imaging of a human inflammatory breast cancer model.

Background: Inflammatory breast cancer (IBC) remains the most aggressive type of breast cancer with the greatest potential for metastasis and as a result, the highest mortality rate. IBC cells invade and metastasize through dermal lymphatic vessels; however, it is unknown how lymphatic drainage patterns change during IBC growth and metastasis. Herein, we non-invasively and longitudinally imaged lymphatics in an animal model of IBC using near-infrared fluorescence (NIRF) imaging.Materials and methods: Mice were imaged in vivo prior to, and up to 11 weeks after subcutaneous or orthotopic inoculation of human IBC SUM149 cells, which were stably transfected with infrared fluorescence protein (iRFP) gene reporter (SUM149-iRFP), following intradermal (i.d.) injection of indocyanine green (ICG).Results: Fluorescence images showed well-defined lymphatic vessels prior to SUM149-iRFP inoculation. However, altered lymphatic drainage patterns including rerouting of lymphatic drainage were detected in mice with SUM149-iRFP, due to lymphatic obstruction of normal lymphatic drainages caused by tumor growth. In addition, we observed tortuous lymphatic vessels and extravasation of ICG-laden lymph in mice with SUM149-iRFP. We also observed increased and dilated fluorescent lymphatic vessels in the tumor periphery, which was confirmed by ex vivo immunohistochemical staining of lymphatic vessels.Conclusions: Our pre-clinical studies demonstrate that non-invasive NIRF imaging can provide a method to assess changes in lymphatic drainage patterns during IBC growth and metastasis.

Abstract P6-12-14: Histone deacetylase inhibitor suberoylanilide hydroxamic acid targets breast cancer stem cells and inhibits metastasis

Abstract Introduction: Inflammatory breast cancer (IBC) is a distinct and aggressive subtype of locally advance breast cancer associated with increased aldehyde dehydrogenase 1 (ALDH1) positive cancer stem cells (CSCs). IBC is associated with a poor survival rate (40% 5-year survival), with few therapeutic strategies identified that effectively blocked the growth and metastasis of IBC. Our previous in vitro studies revealed that the pan-histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) effectively targets self-renewal of IBC tumor cells. Materials and Methods: The IBC cell line, SUM149 was tagged with the Luciferase gene (SUM149-LUC) and their in vivo growth in immune compromised mice tested in both orthotopic and metastatic settings. Results: SUM149-LUC rapidly develop primary tumors and metastatic lesions at a variety of locations including lung, brain, bone, liver, reproductive organs and adrenal glands. SAHA effectively blocked growth of SUM149 primary tumors and inhibited metastasis, which was synergistic with the microtubule stabilizer, Paclitaxel. The therapeutic efficacy of SAHA was associated with a significant decrease in ALDH1+ CSCs populations. Conclusions: Collectively, these results suggest that strategies of combining agents such as SAHA that target CSCs with Paclitaxel that targets the bulk of proliferating tumor cells warrant further investigation for their potential effectiveness in IBC patients, who have the lowest survival of breast cancer patients and have few therapeutic options. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-14.

Abstract P6-12-02: Survival differences between patients with metastatic inflammatory and non-inflammatory breast cancer

Abstract Background: Very little is known about the survival of patients with inflammatory breast cancer (IBC) and distant metastasis. Furthermore, the American Joint Committee on Cancer classification of breast cancer does not recognize metastatic IBC as a distinct entity within stage IV. We hypothesized that the survival of patients with IBC and distant metastasis is worse than the survival of patients with stage-matched non-IBC. Patients and Methods: We retrospectively reviewed 5314 consecutive patients with stage III or IV breast cancer (IBC or non-IBC) who were treated at our institution between 1986 and 2012. A total of 1079 patients presented with IBC (stage III: 861; stage IV: 218) and 4235 non-IBC (stage III: 2781; stage IV: 1454). We compared the time to distant metastasis from initial diagnosis, distant metastasis–free survival (DMFS), and overall survival (OS) in stage-matched patients with IBC or non-IBC. Results: The median follow-up periods were 3.3 years for patients with stage III disease (range, 0-32.2 years) and 1.8 years for patients with stage IV disease (range, 0-19.9 years). The total number of recorded events (metastasis/death) was 1657 for stage III, while the numbers of deaths for stage III and IV were 1337 and 973, respectively. In patients with stage III, the time to distant metastasis was shorter in IBC than in non-IBC (median 1.3 vs. 1.7 years, P < .001). DMFS and OS were shorter in patients with stage III IBC than in those with stage III non-IBC (2.5 vs. 6.9 years, P < .001; and 4.7 vs. 8.9 years, P < .001; respectively). However, there was no significant difference in OS after development of distant metastasis between stage III IBC and non-IBC (median for both 1.3 years, P = .83). In multivariate analysis, the diagnosis of IBC remained significantly associated with mortality after adjusting for potential confounders. De novo stage IV IBC presented more frequently with multiple sites of metastasis than de novo stage IV non-IBC (P = .02). In patients with de novo stage IV disease, OS was shorter in IBC than in non-IBC (2.3 vs. 3.4 years, P = .004). In the multicovariate Cox model, while ethnicity, tumor grade, hormone receptor status and HER2 status, site of metastasis, number of sites of metastasis, and definitive breast surgery by 1 year were all significant factors in OS for stage IV breast cancer, the diagnosis of IBC conferred a hazard ratio of 1.33 (95% confidence interval: 1.05 - 1.69) in multivariate analysis. Conclusion: Our findings suggest that IBC patients with metastasis at diagnosis have worse outcomes than stage-matched non-IBC patients. IBC patients presenting with de novo stage IV disease should be considered as a separate subcategory of stage IV in the tumor-node-metastasis classification because their clinical course and prognosis are different from those of patients with stage IV non-IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-02.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKβ in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 μM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+ IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-497) contains supplementary material, which is available to authorized users.

Abstract P4-06-03: Zinc Finger Nuclease Genome Engineering Reveal Multiple Functions of Secretory Leukocyte Peptidase Inhibitor in Regulating Pleuripotency of Cancer Stem Cells in Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of this disease and is known to be enriched for cells with a cancer stem cell phenotype. IBC is characterized by the presence of cell aggregates, defined as tumor emboli, that metastasize into skin and chest wall. The only documented biomarker of tumor emboli is the surface glycoprotein, E-cadherin. We previously demonstrated that IBC tumor emboli express the alarm anti-protease, secretory leukocyte peptidase inhibitor (SLPI), a metastasis related gene highly expressed in IBC patient tumors. Since the function of SLPI in IBC is unknown, the present studies used zinc finger technology to knockout (KO) copies of SLPI in the SUM149 IBC cell line to define the role of SLPI in IBC. Materials and Methods: Using CompoZr zinc finger nuclease (ZFN) technology (Sigma-Aldrich), SLPI KO cell lines were generated by disrupting all alleles (3) in exon 1 of SUM149 cells derived from an IBC primary tumor with a high CD44+cell population. The target-specific ZFNs bound DNA at a sequence-specific location and created double strand breaks repaired by non-homologous end joining, resulting in deletions at the SLPI locus. Single cell SLPI KO clones were isolated and serially passaged to establish stable cell lines. Functional assays were used to assess proliferation, invasion, tube formation and clonogenicity. Global transcriptional profiling was performed to identify genes and signaling pathways directly regulated by SLPI. Results: SUM149 SLPI KO clones did not produce SLPI protein and had a significantly slower turn-over time of 75 hrs compared with 24 hrs in SUM149 wild type clones. Loss of SLPI blocked invasion by 50%, and completely inhibited formation of tube-like structures, an activity defined as vasculogenic mimicry, characteristic of IBC. The loss of only 1 SLPI allele resulted in the inability of SUM149 cells to grow as anchorage independent clones in soft agar, commonly used as a predictor of in vivo tumorigenicity. SLPI directly regulated expression of multiple genes within the embryonic stem cell pleuripotency canonical pathway, including WNT, Frizzled, PDK-1, platelet derived growth factor receptor and sphingosine-1-phosphate receptor. Studies are underway to determine the specific role of SLPI in IBC tumor growth and metastasis. Conclusions: Our previous studies demonstrated that SLPI is expressed by IBC tumor emboli and can be used as a biomarker of tumor emboli in IBC core and skin punch biopsies. SLPI was found to not only regulate critical functional activities of IBC tumor cells but also to directly regulate genes within pathways critically important to maintenance of pleuripotency of tumors with a cancer stem cell phenotype. Collectively, these studies demonstrate the power and utility of the zinc finger technology, which enables the interrogation of tumor cells to discern the direct function and role of specific genes of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-03.

Differential effects of vitamin D treatment on inflammatory and non-inflammatory breast cancer cell lines

Vitamin D is a known regulator of breast cancer cell proliferation, apoptosis, migration, invasion and differentiation in vitro. Recent studies have suggested a preventative role for vitamin D in breast cancer development and suggested a possible therapeutic application of vitamin D for patients with various forms of breast cancer. Inflammatory breast cancer (IBC) is a highly aggressive and phenotypically unique form of breast cancer that has a very poor prognosis. IBC invades the dermal lymphatics of the breast as tumor emboli early in the course of the disease. Because of the invasive nature of IBC, novel therapeutics are needed desperately. In the current study we examined the effect of the active form of vitamin D, calcitriol, treatment on the aggressive IBC phenotype. Herein we demonstrate that although the vitamin D receptor (VDR) is present in both IBC and non-IBC cell lines, the effect of vitamin D treatment is significant only on the IBC cells. SUM149 IBC cells showed increased protein concentration in response to 24h of calcitriol exposure; likely mediated by an increase in protein synthesis as opposed to increased cellular proliferation. In addition, treatment with 100nM calcitriol showed a significant decrease in SUM149 migration (67.8% decrease, P=0.030), invasion (43.9% decrease, P=0.015), and tumor spheroid size (69.4% decrease, P=0.018) compared to nontreated control groups. Finally, calcitriol treatment of SUM149 cells led to significantly fewer IBC experimental metastases as compared to control. Our study demonstrates that calcitriol treatment of SUM149 affected several of the processes important for IBC metastasis but had little effect on MDA-MB-231 cells. Therefore, calcitriol treatment may have the potential to decrease the rate and incidence of metastasis in IBC patients.

Abstract 569: Targeting inflammatory pathways as an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive type of advanced breast cancer with a poor long-term outcome and a high risk of recurrence. Although one of the most common clinical manifestations of IBC is erythema of the skin overlying the breast, whether inflammation is involved in IBC has not yet been tested by pathologic examination of IBC specimens. Therefore, whether inflammation pathways are involved in the pathogenesis of IBC is still not clear. A large body of evidence indicates that chronic inflammation is a risk factor for cancer. Nonsteroidal anti-inflammatory drugs have been reported to reduce the risk of developing some solid cancers, including breast cancer. Cyclooxygenase (COX)-2 is the inducible isoenzyme catalyzing the conversion of arachidonic acid to prostanoids. COX-2 upregulation has been linked to breast tumorigenesis or inflammatory diseases, and COX-2 overexpression increases the growth rate and invasiveness of breast cancer cells. We tested the hypothesis that the COX-2 pathway is involved in IBC tumor growth and metastasis. Methods and Results: We examined the expression level of COX-2 and the concentrations of its enzymatic products by mass spectrometry (LC/MS/MS). We also determined the effect of inhibition of the COX2 pathway on cell proliferation, migration, invasiveness, and epithelial-mesenchymal transition (EMT) in IBC both in vitro and in vivo. We found that levels of COX-2 and its enzymatic products prostaglandins (PG), such as PGE2 and PGF2α, were higher in IBC than in non-IBC cell lines. Both PGE2 and PGF2α markedly enhanced the migration and invasion of SUM149 IBC cells, suggesting that COX-2 and its metabolites may play an important role in IBC development and progression. We examined the expression levels of COX-2 and EGFR in 25 IBC patient biopsy samples and found that the expression of COX-2 was positively correlated with the levels of EGFR in these tissues. To illustrate how COX-2 and EGFR are regulated in IBC cells, we treated SUM149 cells with EGF. We found that EGF stimulation increased COX-2 levels. COX-2 expression was notably downregulated in EGFR shRNA stably transfected SUM149 cells, suggesting that the upregulation of COX-2 is mediated through EGFR signaling. Furthermore, a selective COX-2 inhibitor, celecoxib, dramatically inhibited IBC tumor growth in a SUM149 IBC xenograft model. These celecoxib-treated tumors exhibited high expression of the epithelial marker E-cadherin and low expression of the mesenchymal marker vimentin compared with untreated tumors, suggesting that celecoxib inhibits epithelial-mesenchymal transition. Conclusions: Our results suggest that inflammatory mediators indeed play an important role in IBC cells. Targeting inflammatory pathways may represent an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in IBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 569. doi:1538-7445.AM2012-569

Targeting of Calcitriol to Inflammatory Breast Cancer Tumors and Metastasis In Vitro and In Vivo

Inflammatory Breast Cancer (IBC) is arguably the most severe form of breast cancer with a very high mortality rate. IBC is distinct from other forms of breast cancer expressing unique cell surface markers such as hypoglycosylated MUC1. However, treatment options for IBC are limited. Calcitriol is a potential treatment for IBC due to its potential role as a therapeutic in other forms of cancer. Our previous research demonstrated that calcitriol inhibits the metastatic ability of the SUM149 IBC cell line. However, high concentration of calcitriol would be required for treatment. This may result I serious side effects such as hypercalcemia. Targeting calcitriol directly to the tumor site would allow for treatment without toxic levels of calcitriol. Here we developed SM3MUC1 antibody conjugated calcitriol bound QDs as a novel nanoparticles probe. We demonstrate that these particles can be used to target to MUC1 over-expressing IBC cells in vitro and in vivo. Therefore these particles can be used to determine the localization of IBC emboli in vivo and maybe used as a potential vehicle to deliver high doses of calcitriol to the IBC tumors and metastasis.

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

Nomogram for predicting development of brain metastasis in patients with inflammatory breast cancer.

243 Background: Inflammatory breast cancer (IBC) is associated with poor prognosis and high rates of distant metastasis. Brain metastasis is particularly important due to its morbidity and mortality. The purpose of this study was to construct a nomogram from clinicopathological factors that could predict the risk of occurrence of brain metastasis in patients with IBC. Methods: We performed a retrospective analysis of 406 patients with IBC, who were seen at the University of Texas M. D. Anderson Cancer Center between 1991 and 2009. Data was collected from IBC registry. Patients were divided in a randomized manner into a training cohort (n = 206) and a validation (n = 200) cohort. A multivariate cox proportional hazards regression analysis of selected prognostic features was performed on the training cohort. The model estimated in the training cohort was cross-validated with the validation cohort. Based on this model a nomogram was constructed to assess 5- and 10-year risk of developing brain metastasis. Results: A total of 115 (28%) patients developed brain metastasis. Univariate analysis identified menopausal status, progesterone receptor expression, number of distant metastasis, adjuvant taxane-based therapy and age at diagnosis as significant factors associated with brain metastasis (p < 0.05). Subsequent multivariate analysis showed menopausal status and number of metastasis were independently associated with development of brain metastasis (p < 0.05). Nomogram constructed using above model was evaluated for its ability to discriminate among likelihood of occurrence of brain metastasis in patients, using the validation set. The validation set showed good discrimination with the area under the receiver operating characteristic curve of 0.68. Conclusions: We have formulated a distinct predictive tool to evaluate 5 and 10-year risk for developing brain metastasis in patients with IBC. This nomogram can be potentially helpful for designing risk adapted therapeutic trials. Identifying patients at high risk for brain metastasis in IBC can help personalize therapy, surveillance, and prognostication. [Table: see text]

Insight into the 6-Thiopurine-Mediated Termination of the Invasive Motility of Tumor Cells Derived from Inflammatory Breast Cancer

Our study showed that a combination of 6-thiopurine (6-TP) drugs and a redox agent effectively inhibits the motility of SUM cells derived from human inflammatory breast cancer (IBC) cells and RhoC-overexpressed mammary epithelium cells. This 6-TP-mediated inhibition of cell motility occurs because the treated 6-TPs target and inactivate RhoC. A molecular mechanism for inactivation by the 6-TP-mediated RhoC is proposed by which treated TPs are converted in cells into 6-thioguanosine phosphate (6-TGNP). This 6-TGNP in turn reacts with the Cys(20) side chain of the redox-sensitive GXXXCGK(S/T)C motif of RhoC to produce a 6-TGNP-RhoC disulfide adduct. A redox agent synergistically enhances the formation process of this disulfide. The adduct that is formed impedes RhoC guanine nucleotide exchange, which populates an inactive RhoC. Our results suggest that 6-TGNP can also react with the redox-sensitive GXXXCGK(S/T)C and GXXXXGK(S/T)C motif of RhoA and Rac, respectively, to produce a 6-TGNP-RhoA and 6-TGNP-Rac disulfide adduct. However, given that RhoC has been shown to be overexpressed in ∼90% of IBC lesions, the populated RhoC but not other Rho proteins is likely to be a primary target for 6-TPs and a redox agent to terminate the metastasis of IBC.

Abstract P5-05-05: Tazarotene-Induced Gene 1(TIG1) Is a Novel Tumorigenic Gene of Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of advanced breast cancer. Effective standard therapies for IBC are limited and the molecular mechanisms underlying IBC are largely unknown. In a comparison of samples from patients with IBC and non-IBC, our laboratory identified TIG1 (tazarotene-induced gene 1), also known as retinoic acid receptor responder 1 (RARRES1) as the gene with the largest difference in expression between the two groups. TIG1 is specifically expressed in IBC cell lines (SUM149 and KPL-4). In addition, TIG1 has been identified as a top druggable gene for triple-negative breast cancer. Of particular interest is that TIG1 is significantly expressed in IBC ER-/HER2- patient samples when compared with non-IBC ER-/HER2- patient samples (p = 0.084).Therefore, we hypothesize that TIG1 plays an important role in the malignant process of IBC and it promotes the tumorigenesis and metastasis of IBC. In these studies we determined the tumorigenic and metastatic activities of TIG1 in IBC. Methods: TIG1 was stably knocked down in the highly TIG1-expressing IBC cell line SUM149 and the effects of knockdown on cell proliferation, migration, invasion, and growth in three-dimensional culture were analyzed in vitro. To evaluate the tumorigenic activity of TIG1 in vivo, TIG1 stable-knockdown SUM149 cells and control shRNA-transfected cells were implanted into the mammary fat pads of athymic nude mice, and tumor growth was monitored. To investigate the underlying mechanism of TIG1- mediated tumorigenesis and metastasis of IBC, the gene expression profiles of control siRNA-transfected and TIG1 siRNA-transfected SUM149 cells were compared using cDNA microarray analysis. Results: Suppressing TIG1 significantly inhibited the proliferation, migration, and invasion of IBC cells in vitro. SUM149 cells exhibit a mesenchymal-like phenotype when grown in three-dimensional culture. However, knocking down TIG1 reversed the mesenchymal-like phenotype to the epithelial phenotype in three-dimensional culture, suggesting that TIG1 might have metastatic activity in IBC cells. TIG1 knockdown dramatically inhibited IBC tumor growth in a xenograft model. Several differentially expressed genes between control siRNA-transfected and TIG1 siRNA-transfected SUM149 cells, including Decorin, GBP1, and VAV3 were identified using cDNA microarray analysis. These genes have been reported to affect a number of biological processes, including proliferation, metastasis, inflammatory response, and angiogenesis. We will focus on these candidates and determine the molecular mechanism by which TIG1 may function as a tumorigenic gene in IBC. Conclusion: Our data demonstrate that TIG1 promotes the tumorigenesis and metastasis of IBC and it may contribute to the development and aggressiveness of IBC. Our long-term goal is to develop TIG1 as a therapeutic target for this extremely aggressive type of breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-05.